This U19 Cooperative Agreement grant application is designed to perform preclinical studies to optimize a combination microbicide composed of the HIV-1 entry inhibitor retrocyclin RC101 and the nonnucleoside reverse transcriptase inhibitor (NNRTI) 5-chloro-3-phenylsulfonylindole-2-carboxamide (CSIC). Our goal is to develop a microbicide that will have potent anti-HIV activity against cell-free and cell-associated virus while inducing minimal viral resistance, be non-toxic and non-inflammatory to cervical and vaginal tissues and be safe and effective against vaginal SHIV challenge in monkeys. An optimal combination of RC-101 and CSIC with these properties will be chosen based on a highly interactive approach between four projects and two cores. Together, we will employ an array of in vitro and ex vivo laboratory tests of efficacy and toxicity, in vivo safety and efficacy tests in monkey models, and studies to optimize a silicone elastomer ring formulation of our compounds. The 4 projects of this grant application are: 1) Development of CSIC as a microbicide (Project 1, Dr. Parniak), 2) RC-101 as an intravaginal anti-HIV-1 topical microbicide (Project 2, Dr. Cole), 3) Testing candidate microbicides for antiviral activity and toxicity in a cervical tissue based organ culture (Project 3, Dr. Gupta), and 4) Evaluation of toxicity and efficacy of CSIC and RC-101 in monkeys (Project 4, Dr. Marx). The scientific studies in these four projects will be supported by the Administrative Core (Core A, Dr. Gupta) and a Formulation Core (Core B, Drs. Rohan and Smith). Dr. Tom Smith (Auritec Pharmaceuticals), serving as the Industry partner in this U19 program, will bring controlled release pellet technologies to develop vaginal ring delivery of RC-101- and CSIC-based microbicides in monkeys. In designing the scope of the proposed topical microbicide program, we have focused on extensively evaluating safety and toxicity of candidate microbicides utilizing in vitro studies, tissue explants and animal models, as well as an expanded and innovative organ culture model to study the antiviral activity of microbicides in the presence of common STIs. Our multidisciplinary approach to microbicide development targets two different sites of HIV-1 replication (entry and reverse transcription) through a controlled-release ring formulated product, utilizes a novel ex vivo organ culture to test toxicity, inflammation and antiviral activity across cervicovaginal mucosa, and applies macaque models to study toxicity and efficacy. Therefore, the proposed study will provide novel strategies to develop microbicides for the prevention of HIV-1 transmission and their sequelae for women. PROJECT 1: Development of CSIC as a Microbicide (Parniak, M) PROJECT 1 DESCRIPTION (provided by applicant): Anti-HIV topical microbicides are an accessible means to minimize HIV transmission. Certain HIV reverse transcriptase inhibitors (RTIs) are promising microbicide candidates and microbicides based on NNRTI's (UC781 and TMC120) as well as one with a nucleotide RT inhibitor (PMPA;tenofovir) are in Phase 1 clinical trials. However, there is a definite need to identify new pipeline RTI's as backup microbicidal agents as it is well known that many drugs with promising preclinical properties fail during advanced clinical evaluation. The novel NNRTI 5-chloro-3-(phenylsulfonyl)indole-2-carboxamide (CSIC) may represent an important pipeline drug as our preliminary data suggest that the in vitro microbicidal efficacy of CSIC is as good or superior to that of UC781. Combination microbicides directed at different HIV targets may be preferable and we propose that the combination of CSIC and the HIV entry inhibitor antimicrobial peptide RC-101 will provide superior broad spectrum anti-HIV microbicidal activity. In this context, we propose the following Specific Aims for this Program Project component: (1) To evaluate the in vitro microbicidal properties of CSIC alone and in combination with RC-101;(2) To determine the mechanism of the CSIC-induced protective or "memory" effect;(3) To determine whether microbicides based on CSIC alone and in combination with RC-101 will select in vitro for transmission of NNRTI-resistant virus;and (4)To develop and validate an analytical method to quantify the plasma, cellular and tissue levels of CSIC following vaginal or rectal topical administration in monkeys (Project 4).